Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2300RC, Leiden, The Netherlands.
Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2300RC, Leiden, The Netherlands; Department of Pathology, Leiden University Medical Center, Albinusdreef 2, 2300RC, Leiden, The Netherlands.
Anal Chim Acta. 2017 Aug 8;980:1-24. doi: 10.1016/j.aca.2017.05.011. Epub 2017 May 24.
Metabolomics analysis of body fluids as well as cells is depended on many factors. While several well-accepted standard operating procedures for the analysis of body fluids are available, the NMR based quantitative analysis of cellular metabolites is less well standardized. Experimental designs depend on the cell type, the quenching protocol and the applied post-acquisition workflow. Here, we provide a tutorial for the quantitative description of the metabolic phenotype of mammalian cells using NMR spectroscopy. We discuss all key steps of the process, starting from the selection of the appropriate culture medium, quenching techniques to arrest metabolism in a reproducible manner, the extraction of the intracellular components and the profiling of the culture medium. NMR data acquisition and methods for both qualitative and quantitative analysis are also provided. The suggested methods cover experiments for adherent cells and cells in suspension. We ultimately describe the application of the discussed workflow to a thyroid cancer cell line. Although this tutorial focuses on mammalian cells, the given guidelines and procedures may be adjusted for the analysis of other cell types.
体液和细胞的代谢组学分析取决于许多因素。虽然有几种被广泛认可的体液分析标准操作规程，但基于 NMR 的细胞代谢物定量分析的标准化程度较低。实验设计取决于细胞类型、淬灭方案和应用的采集后工作流程。在这里，我们提供了一个使用 NMR 光谱法定量描述哺乳动物细胞代谢表型的教程。我们讨论了该过程的所有关键步骤，从选择合适的培养基、以可重复的方式停止代谢的淬灭技术开始，到细胞内成分的提取和培养基的分析。还提供了 NMR 数据采集和定性及定量分析的方法。所建议的方法涵盖了贴壁细胞和悬浮细胞的实验。我们最终描述了所讨论的工作流程在甲状腺癌细胞系中的应用。尽管本教程侧重于哺乳动物细胞，但给出的指南和程序可以根据其他细胞类型的分析进行调整。
